The diterpenoid, salvinorin A is a potent psychoactive component of the indigenous Mexican plant Salvia divinorum used medicinally by the Mazatec Indians for treating headaches, arthritis and anaemia (Valdes et al. I983). The CNS activity of this normitrogenous molecule is attributed to its strong affinity for the kappa-opiod receptor (KOR) (Roth et al 2002). Its use as a recreational drug is increasing due to its ready availability and potent activity. Ingestion of S. divinorum or salvinorin A (from here-on, reference to salvinorin A use or ingestion implicitly includes S. divinorum) is usually by way of leaf chewing, intake of a liquid extract of the leaves or smoke inhalation. Its CNS effects and hallucinogenic properties have been compared to LSD, leading to its illegal status in many countries. The KOR has been implicated in nociception and a number of disease processes, and there is great interest in the active ingredient of S. divinorzam, salvinorin A (and its analogues) as a potential treatment for various conditions including diarrhoea, mood disorders, and in the regulation of pain (Vortherms and Roth, 2006). The use of salvinorin A and analogues as a potential treatment for mania is described in WO 2005/089745; US 2006/0058264 describes salvinorin A and analogues as useful compounds for pharmacological research purposes and for disease treatment; US 2006/0083679 proposes the use of salvinorin A and analogues as medicines or as chemical probes in diagnostic procedures such as PET, SPECT and NMR spectroscopy. This interest has resulted in the synthesis and pharmacological study of various salvinorin analogues. More recently, C-9 ether derivatives of salvinorin A have been shown to be more active and have a greater half-life than salvinorin A. This development has generated interest in the scientific research community regarding potential new therapeutic drugs, although the broader societal consequences of the abuse of such potent CNS-active molecules are unknown.
The short action of duration of salvinorin A (approximately 10-15 minutes) suggests rapid metabolism to an inactive form. Phamacokinetic studies by Hooker et al (2008) using PET supported the rapid uptake and short duration of action of salvinorin A. The C-9 hydroxylated analogue, salvinorin B, is speculated to be the main metabolite formed by esterase-mediated hydrolysis (Yan and Roth 2004; Schmidt et al 2005a). Studies by Schmidt et al (2005a, 2005b) on monkey plasma were inconclusive as the ex vivo study identified salvinorin B as a metabolite, while the in vivo study did not detect salvinorin B. Pichini et al (2005) were unable to detect salvinorin A in the saliva, sweat or urine of patients 1.5 hours after they had smoked the drug, suggesting either rapid elimination or extensive metabolism. Tsujikawa et al (2009), in an in vitro metabolic study using rat plasma, identified salvinorin B and 1,4a-dimethyl-l-[2-(3-furanyl)-2-hydroxyethyl]-7-hydroxy-5-methoxycarbonyl-8-oxodecahydronapthalene-2-carboxylic acid as the main metabolites. Thus, although there is mounting evidence that salvinorin B is the main metabolite of salvinorin A, besides the metabolites proposed by Tsujikawa, there are likely to be as yet unidentified metabolites. A comparative analysis of the metabolic pathway of other psychoactive drugs with similar molecular structures, methods of ingestion and pharmacological properties enables predictions of other possible salvinorin A metabolites. Drug metabolism usually involves the formation of more polar substances to facilitate excretion. This occurs through first-phase (blood-based) oxidation mediated by cytochrome P450 enzymes and second-phase (liver-based) glucuronidation. The structural and pharmacological similarities of salvinorin A and Δ9-THC, as well as their similar methods of ingestion, suggests metabolic data derived from Δ9-THC research might be a useful indicator of the likely metabolic products of salvinorin A. One metabolite of Δ9-THC is the primary alcohol 11-hydroxy-Δ9-THC, formed through oxidation of the methyl group attached to the alkene group of the heterocyclic system. The primary alcohol is further oxidised to the main metabolite of Δ9-THC, 11-nor-Δ9-THC-9-carboxylic acid which also undergoes glucuronidation. Cocaine and heroin, potent CNS-acting drugs, like salvinorin A, each possess two ester functionalities and the metabolic pathways of the three drugs might be expected to show similarities. Cocaine and heroine are metabolized by human carboaylesterases (hCEs), enzymes expressed in various organs including the liver, intestines and lungs (Imai et al 2006}. Based on comparative scientific evidence and pharmacological properties of salvinorin A and its analogues, salvinorin A-7-carboxylic acid and salvinorin B-7-carboxylic acid are potential metabolites of salvinorin A.
Several analytical methods have been devised to detect and quantify salvinorin A and its analogues in S. divinorum (Grundmann et al 2007). Besides the rat plasma study of Tsujikawa, detection in animal fluids has been limited to the detection of salvinorin A and salvinorin B. Schmidt et al (2005a) used HPLC-MS to detect salvinorin A and salvinorin B in monkey plasma samples ex vivo. They reported that salvinorin B could not be clearly detected in in vivo samples. Pichini et al (2005) used GC-MS to analyse the urine, saliva and sweat of two individuals who had smoked S. divinorum leaves. As previously described, salvinorin A was detected in urine within the first 1.5 hours (limit of detection was 5 ng/ml). After 1.5 hours the technique did not detect salvinorin A in either urine or saliva, probably limited by the sensitivity of the assay. McDonough et al (2008) developed a HPLC-MS to detect and quantitate salvinorin A in human biological fluids stating previously described methods as irreproducible. The method had a limit of detection of 2.5 ng/ml and a limit of quantitation of 5.0 ng/ml. They also suggested that due to rapid disappearance of salvinorin A, identification of a metabolite of the primary active substance is desirable.
To enable a robust detection method which identifies the use of salvinorin A, provision must be made to detect not only recognised markers such as the parent salvinorin A and C-9 metabolite salvinorin B, but also, as yet unidentified metabolites. In addition this method must be highly sensitive to salvinorin A, the one unequivocal marker of salvinorin A use. A highly sensitive and relatively simple method for detecting the ingestion of salvinorin A by the in vitro analysis of human biological samples has so far not been developed. Furthermore, therapeutic research has already identified, and will continue to identify, highly active analogues of salvinorin A which the recreational drug taker will exploit.
It is evident that existing assay formats for the detection of salvinorin A are inadequate, and do not enable such robust detection. Know detection methods use expensive equipment which have low sensitivity for salvinorin A and hence a limited window of detection following salvinorin A ingestion. Furthermore, these assays do not address detection of new and future synthetic analogues of greater stability and activity than salvinorin A. Thus methods are also required to detect the next generation of salvinorin A based drugs.